Expression, Purification, and Characterization of bacterial protein Introduction:
Recombinant proteins were [are] used throughout biomedicinal science.[1] Specifically, most of the proteins were generally suitable for expression in E-Coli bacteria. [2] Throughout many years, many researchers were trying to optimize E-Coli as an expression host for proteins from higher organisms, such as many human integral membrane proteins. [2] The goal of the first part of the experiment was to transform competent bacterial cells with a DNA plasmid and to inoculate a larger culture and express the recombinant protein. By controlling the ratio of recombinant protein to the column size, the final purity of the protein could be optimized.[2] by providing the imidazole in chromatography buffers, lower-affinity contaminants could be competed with an excess of histidine-tagged recombinant protein.[2] Second goal of this experiment was to break open the bacterial cells to release the soluble proteins and purify the protein using the affinity tag and Ni-NTA resin. Characterizing the protein lessen the risk of wasting resources on protein material. [2] After the purification, protein samples were resolved by denaturing SDS-PAGE and this method could also separate proteins from the samples. The final goal of this experiment was to use the electrophoresis and spectroscopy results to estimate the molecular weight of the gbr22 protein as well as to purify and yield of the final purified protein product. The purpose of this lab was to apply the techniques of protein expression, purification, and characterization as well as to understand the fundamental mechanisms in recombinant protein of bacteria and applications to targeting human diseases, which was the essential to virtual drug screening. The hypothesis of this experiment was that those techniques could allow a researcher to successfully estimate the molecular weight of gbr22 protein in the final purified protein product.
Materials & Methods:
The materials for the whole experiment included ice bucket, 42 Celsius water bath, gas burner, 3 1x14 ml round bottom transformation tubes, water, 37 Celsius incubator, colirollers, 2LB Agar Amp plates, 1 spare Agar plate with antibiotic, competent cells, plasmid DNA, LB media, SOC media, pipettes. 1M Imidazole, 1xPBS, 1.7ml centrifuge tubes, 2x10ml round bottom tubes, 4x15ml conical tubes, ring stand with clamps, Ni-NTA resin, Cyanase, and Bio-Rad Econo chromatography column with yellow cap and clear round top. Furthermore, materials include heat block at 95 Celsius, Mini-Protein electrophoresis tank and lid, TGS running buffer, Bio-Rad precast polyacrylamide gel, 6x gel loading buffer, plastic container, Imperial protein stain, and Molecular Weight Standard.
In the protein expression part, 25 µl of E-coli BL21 bacteria was added to each of the 2 transformation tubes, and 1-2 µl pGEM-gbr22 plasmid was added to bacteria in the DNA tubes; control tube had no DNA. After 30 minutes of waiting time on ice, tubes were heat shocked in 42 Celsius water bath for 45 seconds. After another 2 minutes, 200 µl of SOC media was added and the mixture was shake in the incubator for 30 minutes at 37 Celsius. Then 5-6 colirollers were placed onto each plate and 50 µl of bacteria/SOC mixture was pipetted from the tube onto each plate. There was an additional 'fun plate' which mixed dusts in a lab corner and was placed inside of the LB culture. On Day2, sterile pipette tip was used to pick a single colony of bacteria growing on the LB plate. The selected colony was transferred into LB, and then mixed ampicillin to fresh LB to 100µl/ml and transfer the previous culture into the amp/LB solution in the flask, the mixture in the shaking incubator was secured. On Day4, tubes were centrifuged at 5000rpm and pellet’s weight was obtained. Stock lysozyme was added to mix into 1mg/ml.
In the protein purification, 2µl of Cyanase was added into the tubes and then centrifuged at 14000rpm, then the lysate was syringe filtered. Batch and column chromatography was applied in Ni-TNA affinity purification, and the elution1 and 2 were obtained. Nanodrop spectrophotometer was used to measure the elution1 at 280nm and maximum wavelength. The concentration of the protein and yield were determined.
In the protein characterization, 6x loading buffers were added into samples1-6 and the samples were heat blocked at 95 Celsius for 5 minutes. TGS buffer was syringed into each well of the SDS-PAGE, and then 7µl of each sample was pipetted into each well, after running 25 minutes at 200V, the gel was collected. Imperial protein stain was mixed with the gel and was shaked in the orbital shaker. On day2, gel was vacuum pumped and results were obtained.
Results:
Figure 1: The control plate with Ampicillin and BL21 (DE3) and no DNA. No colonies were displayed.
Figure 2: The experimental plate with Ampicillin and BL21 (DE3) and DNA. Many colonies of BL21 (DE3) E.Coli were displayed.
Figure 3: Fun Plate with no Ampicillin or BL21 (DE3) or DNA. The source was from a swipe of the corner of the window mixed with agar.
Figure 4: The flask contains LB, ampicillin, BL21 and pGEM-gbr22. The liquid displayed purple. The bacteria had successfully grown.
Figure 5: The centrifuged Cell pellet (0.4g) contained BL21 and pGEM-gbr22 in the conical tube.
Figure 6: The 15 ml conical tube containing the elution buffer after the initial wash.
Figure 7: The 15 ml conical tube containing the elution buffer after the second wash.
Figure 8: The absorbance of elution1 at wavelength of 280nm in trail 1 was 0.14.
Figure 9: The absorbance of elution1 at wavelength of 280nm in trail 1 was 0.16.
A = ebc (0.157 + 0.141)/2 = (1cm)x(38850)x(c) c = 3.835x10^-6 mol/L (mol/L)x(g/mol) = mg/ml (3.835x10^-6 mol/L) x (25794.2 g / 1 mol) x (1 L / 1000 ml) x(1000 mg / 1 g) = 0.099mg/ml
Figure 10: The absorbance of elution1 at maximum wavelength of 574nm in trail 1 was 0.22.
Figure 11: The absorbance of elution1 at maximum wavelength of 574nm in trail 2 was 0.23.
A = ebc
(0.25 + 0.21)/2 = (1cm)x(118300)x(c)
c = 1.944x10^-6 mol/L (1.944x10^-6 mol/L) x (25794.2 g / 1 mol) x (1 L / 1000 ml) x(1000 mg / 1 g) = 0.050mg/ml
Figure 12: The gel after placed in the vacuum pump in gradient cycle for 1.5 hours at 75 Celsius temperature.
Figure 13: The molecular Weight Standard, used to estimate the weight of the protein.
Discussion:
In the process of protein expression, the DNA plasmid was transformed into the competent bacterial cells and protein was expressed. The experimental plate with Ampicillin and BL21(DE3) and DNA exhibit the growth of many colonies of E-Coli. The control plate and the ‘fun plate’ had no grown bacteria. The control plate did not have grown bacteria because there was no plasmid inside of the bacterial cell, which had no resistance to ampicillin; this led to no grown of any bacteria. The heat shock process in this experiment was important because this allowed the plasmid to get into the cells of bacteria. Lysozyme was critical in breaking the cell walls of the bacteria, which further facilitated the passage of the plasmid’s path into the cell for purification. There was an accidental spill of the bacterial liquid mix during the experiment, this could lead to contamination of some of the working stations, this could be avoided with proper caution in the future.
In the process of protein purification, bacterial cells were broken and the soluble proteins were successfully released. The protein was successfully purified using the affinity tag and Ni-NTA resin. Lysozyme was important in breaking the cell walls of the bacteria, and Cyanase was important in digesting the DNA in the cells. Elution1 had higher concentration of protein than Elution2. This is because during the elution process, the Imidazole in the elution buffer bound with nickel and kicked off the protein with the histidine, which allowed protein to be filtrated out. Wash buffer had significantly less imidazole than elution buffer, which also explained the much less amount of protein in elution2 than elution1. There were error and difficulty in this process, also. For example, during the purification process, it was hard to keep the protein mixture not to spill over the ice bucket because the process required a fast switch motion of capping. Therefore the results could be influenced because there was less amount of mixture.
In protein characterization, sample1-6 contained proteins and loading buffers after the preparation. On the image of gel, starting with band#5 was the clearest to determine the weight of the protein. The weight was 25kDa when comparing it to the Molecular Weight Standard. The determined molecular weight of gbr22 in process of purification was 25794.2Da, which was very close to the 25kDa, the experimental value. The purity of the sample was 50%. There was a significant error in this part of the lab. During the process of washing the gel, the gel was broken, which could impact the result greatly because some of the information was omitted.
Conclusions:
Throughout the process of protein expression, purification, and characterization, molecular weight of gbr22 protein in the final purified protein product was successfully determined. The next step was the protein assay. By applying those techniques in protein and bacteria, which were essential to the virtual drug screening, the investigator could better understand the fundamentals of recombinant protein in bacteria and perform many similar experiments targeting many human diseases in the future.
References:
[1] François Baneyx, Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology 1999, Volume 10, (Issue5), pp 411-421.
[2] Structural Genomics Consortium, Architecture et Fonction des Macromolécules Biologiques, Protein production and purification. Nat Method 2008, Volume 5, (2), pp 135-46.
Title:
Expression, Purification, and Characterization of bacterial proteinIntroduction:
Recombinant proteins were [are] used throughout biomedicinal science.[1] Specifically, most of the proteins were generally suitable for expression in E-Coli bacteria. [2] Throughout many years, many researchers were trying to optimize E-Coli as an expression host for proteins from higher organisms, such as many human integral membrane proteins. [2] The goal of the first part of the experiment was to transform competent bacterial cells with a DNA plasmid and to inoculate a larger culture and express the recombinant protein. By controlling the ratio of recombinant protein to the column size, the final purity of the protein could be optimized.[2] by providing the imidazole in chromatography buffers, lower-affinity contaminants could be competed with an excess of histidine-tagged recombinant protein.[2] Second goal of this experiment was to break open the bacterial cells to release the soluble proteins and purify the protein using the affinity tag and Ni-NTA resin. Characterizing the protein lessen the risk of wasting resources on protein material. [2] After the purification, protein samples were resolved by denaturing SDS-PAGE and this method could also separate proteins from the samples. The final goal of this experiment was to use the electrophoresis and spectroscopy results to estimate the molecular weight of the gbr22 protein as well as to purify and yield of the final purified protein product. The purpose of this lab was to apply the techniques of protein expression, purification, and characterization as well as to understand the fundamental mechanisms in recombinant protein of bacteria and applications to targeting human diseases, which was the essential to virtual drug screening. The hypothesis of this experiment was that those techniques could allow a researcher to successfully estimate the molecular weight of gbr22 protein in the final purified protein product.
Materials & Methods:
The materials for the whole experiment included ice bucket, 42 Celsius water bath, gas burner, 3 1x14 ml round bottom transformation tubes, water, 37 Celsius incubator, colirollers, 2LB Agar Amp plates, 1 spare Agar plate with antibiotic, competent cells, plasmid DNA, LB media, SOC media, pipettes. 1M Imidazole, 1xPBS, 1.7ml centrifuge tubes, 2x10ml round bottom tubes, 4x15ml conical tubes, ring stand with clamps, Ni-NTA resin, Cyanase, and Bio-Rad Econo chromatography column with yellow cap and clear round top. Furthermore, materials include heat block at 95 Celsius, Mini-Protein electrophoresis tank and lid, TGS running buffer, Bio-Rad precast polyacrylamide gel, 6x gel loading buffer, plastic container, Imperial protein stain, and Molecular Weight Standard.
In the protein expression part, 25 µl of E-coli BL21 bacteria was added to each of the 2 transformation tubes, and 1-2 µl pGEM-gbr22 plasmid was added to bacteria in the DNA tubes; control tube had no DNA. After 30 minutes of waiting time on ice, tubes were heat shocked in 42 Celsius water bath for 45 seconds. After another 2 minutes, 200 µl of SOC media was added and the mixture was shake in the incubator for 30 minutes at 37 Celsius. Then 5-6 colirollers were placed onto each plate and 50 µl of bacteria/SOC mixture was pipetted from the tube onto each plate. There was an additional 'fun plate' which mixed dusts in a lab corner and was placed inside of the LB culture. On Day2, sterile pipette tip was used to pick a single colony of bacteria growing on the LB plate. The selected colony was transferred into LB, and then mixed ampicillin to fresh LB to 100µl/ml and transfer the previous culture into the amp/LB solution in the flask, the mixture in the shaking incubator was secured. On Day4, tubes were centrifuged at 5000rpm and pellet’s weight was obtained. Stock lysozyme was added to mix into 1mg/ml.
In the protein purification, 2µl of Cyanase was added into the tubes and then centrifuged at 14000rpm, then the lysate was syringe filtered. Batch and column chromatography was applied in Ni-TNA affinity purification, and the elution1 and 2 were obtained. Nanodrop spectrophotometer was used to measure the elution1 at 280nm and maximum wavelength. The concentration of the protein and yield were determined.
In the protein characterization, 6x loading buffers were added into samples1-6 and the samples were heat blocked at 95 Celsius for 5 minutes. TGS buffer was syringed into each well of the SDS-PAGE, and then 7µl of each sample was pipetted into each well, after running 25 minutes at 200V, the gel was collected. Imperial protein stain was mixed with the gel and was shaked in the orbital shaker. On day2, gel was vacuum pumped and results were obtained.
Results:
Figure 1: The control plate with Ampicillin and BL21 (DE3) and no DNA. No colonies were displayed.
Figure 2: The experimental plate with Ampicillin and BL21 (DE3) and DNA. Many colonies of BL21 (DE3) E.Coli were displayed.
Figure 3: Fun Plate with no Ampicillin or BL21 (DE3) or DNA. The source was from a swipe of the corner of the window mixed with agar.
Figure 4: The flask contains LB, ampicillin, BL21 and pGEM-gbr22. The liquid displayed purple. The bacteria had successfully grown.
Figure 5: The centrifuged Cell pellet (0.4g) contained BL21 and pGEM-gbr22 in the conical tube.
Figure 6: The 15 ml conical tube containing the elution buffer after the initial wash.
Figure 7: The 15 ml conical tube containing the elution buffer after the second wash.
Figure 8: The absorbance of elution1 at wavelength of 280nm in trail 1 was 0.14.
Figure 9: The absorbance of elution1 at wavelength of 280nm in trail 1 was 0.16.
A = ebc
(0.157 + 0.141)/2 = (1cm)x(38850)x(c)
c = 3.835x10^-6 mol/L (mol/L)x(g/mol) = mg/ml
(3.835x10^-6 mol/L) x (25794.2 g / 1 mol) x (1 L / 1000 ml) x(1000 mg / 1 g) = 0.099mg/ml
Figure 10: The absorbance of elution1 at maximum wavelength of 574nm in trail 1 was 0.22.
Figure 11: The absorbance of elution1 at maximum wavelength of 574nm in trail 2 was 0.23.
A = ebc
(0.25 + 0.21)/2 = (1cm)x(118300)x(c)
c = 1.944x10^-6 mol/L
(1.944x10^-6 mol/L) x (25794.2 g / 1 mol) x (1 L / 1000 ml) x(1000 mg / 1 g) = 0.050mg/ml
Figure 12: The gel after placed in the vacuum pump in gradient cycle for 1.5 hours at 75 Celsius temperature.
Figure 13: The molecular Weight Standard, used to estimate the weight of the protein.
Discussion:
In the process of protein expression, the DNA plasmid was transformed into the competent bacterial cells and protein was expressed. The experimental plate with Ampicillin and BL21(DE3) and DNA exhibit the growth of many colonies of E-Coli. The control plate and the ‘fun plate’ had no grown bacteria. The control plate did not have grown bacteria because there was no plasmid inside of the bacterial cell, which had no resistance to ampicillin; this led to no grown of any bacteria. The heat shock process in this experiment was important because this allowed the plasmid to get into the cells of bacteria. Lysozyme was critical in breaking the cell walls of the bacteria, which further facilitated the passage of the plasmid’s path into the cell for purification. There was an accidental spill of the bacterial liquid mix during the experiment, this could lead to contamination of some of the working stations, this could be avoided with proper caution in the future.
In the process of protein purification, bacterial cells were broken and the soluble proteins were successfully released. The protein was successfully purified using the affinity tag and Ni-NTA resin. Lysozyme was important in breaking the cell walls of the bacteria, and Cyanase was important in digesting the DNA in the cells. Elution1 had higher concentration of protein than Elution2. This is because during the elution process, the Imidazole in the elution buffer bound with nickel and kicked off the protein with the histidine, which allowed protein to be filtrated out. Wash buffer had significantly less imidazole than elution buffer, which also explained the much less amount of protein in elution2 than elution1. There were error and difficulty in this process, also. For example, during the purification process, it was hard to keep the protein mixture not to spill over the ice bucket because the process required a fast switch motion of capping. Therefore the results could be influenced because there was less amount of mixture.
In protein characterization, sample1-6 contained proteins and loading buffers after the preparation. On the image of gel, starting with band#5 was the clearest to determine the weight of the protein. The weight was 25kDa when comparing it to the Molecular Weight Standard. The determined molecular weight of gbr22 in process of purification was 25794.2Da, which was very close to the 25kDa, the experimental value. The purity of the sample was 50%. There was a significant error in this part of the lab. During the process of washing the gel, the gel was broken, which could impact the result greatly because some of the information was omitted.
Conclusions:
Throughout the process of protein expression, purification, and characterization, molecular weight of gbr22 protein in the final purified protein product was successfully determined. The next step was the protein assay. By applying those techniques in protein and bacteria, which were essential to the virtual drug screening, the investigator could better understand the fundamentals of recombinant protein in bacteria and perform many similar experiments targeting many human diseases in the future.
References:
[1] François Baneyx, Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology 1999, Volume 10, (Issue5), pp 411-421.
[2] Structural Genomics Consortium, Architecture et Fonction des Macromolécules Biologiques, Protein production and purification. Nat Method 2008, Volume 5, (2), pp 135-46.